1. Field of the Invention
The present invention relates to separation of macromolecules (e.g., proteins and nucleic acids) via electrophoresis.
2. Description of Related Art
Polymer hydrogels have played an instrumental role in the separation of both proteins and DNA, as these materials are needed for use in medical diagnostics, drug purification, and other scientific research (see, e.g., Sukuru, Karunakar, “Lipophilic Vehicle-Based Dual Controlled Release Matrix System”; U.S. Patent App. Pub. No. 2007/0092560, filed 26 Oct. 2006; and Wilson Moya, Jad, Jaber, “Purification of Proteins” PCT App. Pub No. WO2008079280, filed 20 Dec. 2007, each of which is hereby incorporated by reference in its entirety). These separations are performed using an applied electrical field in a process known as electrophoresis (see, e.g., Probstein, Robert. Physicochemical Hydrodynamics: An Introduction. 2d ed, hereby incorporated by reference in its entirety). Electrophoresis is a separation technique for separating proteins or nucleic acids on the basis of both their charge density and their molecular weight, and is often performed in a hydrogel matrix. Two transport parameters determine these molecular level characteristics: the electrophoretic mobility; and the effective molecular diffusivity. The results of the separation are often termed “fuzzy” meaning that the process does not effectively or sufficiently separate or resolve the proteins in such a mixture. The problem, then, is one of obtaining better resolution and better separation in such cases. Many techniques are currently used to improve separation efficiency. One of the most promising approaches is to modify the morphology of the hydrogel matrix. In using a polymeric gel, there are five major experimental variables one might consider to produce a more complete separation. One can change, for example, the concentration ratio of the matrix monomer and its cross-linker (which affects the porosity of the matrix), the pH and amount of the buffer that makes up the fluid phase, the system temperature, the electrical field applied to the solute, and the shear applied to the system. Adjustment of these variables can improve separation between two solutes based on both size differences (size selectivity or molecular sieving) and charge differences, which will modify either or both the electrophoretic mobility and/or the molecular diffusivity.
The introduction of nanotechnology has recently contributed improvements to electrophoretic separation techniques. By using nano-templates, molecules that segregate particular morphological structures during gel synthesis, and removing these templates post synthesis, new gel morphologies can be created. In Rill, et al's work, both DNA seed molecules as well as Sodium Dodecyl Sulfate (SDS) were used as templates (see, e.g., Dharia J R, Pill R, Van Winkle D, Locke B R, and Arce P, “Preparation and characterization of polyacrylamide gels containing microchannels.” American Chemical Society Annual Meeting, Orlando, Fla., May 5-7, 1994; and Rill R, Locke B R, Liu Y, Dharia J, and Van Winkle D, “Protein electrophoresis in polyacrylamide gels with templated pores.” Electrophoresis, 1996; 17(8):1304-1312, each of which is hereby incorporated by reference in its entirety). The reported results of that work suggested that separation efficiency could be improved in these gels templated with SDS. Trinh et. al used idealized geometric models to simulate Rill's results (S. Trinh, B. R. Locke, and P. Arce, “Diffusive-convective and diffusive-electroconvective transport in non-uniform channels with application to macromolecular separations,” Separation and Purification Technology, vol. 15, pp. 255-269, 1999, hereby incorporated by reference in its entirety). The results of Trinh's study suggest that the morphology of the gel plays an important role in modifying transport of macromolecules and improving electrophoretic separations.
More recently, nanoparticle insertion into hydrogels proved useful when regular, unmodified polyacrylamide gels failed (Huang Guangming, Zhang Yangjun, Ouyang Jin, Baeyens Willy R. G. Delanghe Joris R. “Application of carbon nanotube-matrix assistant native polyacrylamide gel electrophoresis to the separation of apolipoprotein A-I and complement C3.” Analytica Chimica Acta. 557. 2006. 137-145, hereby incorporated by reference in its entirety). In addition, Matos et. al, has reported important changes in electrokinetic based fluxes across modified gels with silica-based nanoparticle insertion (Matos M, Tilton R and White L. “Electroosmotically enhanced mass transfer through polyacrylamide gels.” Journal of Colloids and Inferface Science. Volume 300 Issue 1. Aug. 1, 2006. 429-436, hereby incorporated by reference in its entirety). These studies collectively, show that gel structure can effectively be modified by adding a third component: nanoparticles.
None of the hydrogels in the prior art, however, teach a composite hydrogel matrix comprising a thermoresponsive component in combination with electrophoretic transport. Neither do any hydrogels of the prior art teach a gel therein the matrix morphology may be modified selectively and/or regionally.
The technical problem underlying the present invention was therefore to overcome these prior art difficulties by creating a tunable composite gel that would be effective and useful for electrophoretic studies of solute mobility in narrow channel/wide channel geometry/matrix morphology. The solution to this technical problem is provided by the embodiments characterized in the claims.